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Vaccine 28 (2010) 8306–8314

Contents lists available at ScienceDirect

Vaccine

journa l homepage: www.e lsev ier .com/ locate /vacc ine

CPP (poly[di(carboxylatophenoxy)-phosphazene]) microparticleso-encapsulating ovalbumin and CpG oligo-deoxynucleotides are potentnhancers of antigen specific Th1 immune responses in mice

rinivas Garlapati a,1, Nelson F. Enga,1, Heather L. Wilsona,1, Rachelle Buchanana,eorge K. Mutwiri a,c, Lorne A. Babiukd, Volker Gerdtsa,b,∗

Vaccine and Infectious Disease Organization (VIDO), University of Saskatchewan, 120 Veterinary Road, Saskatoon S7N 5E3, CanadaDepartment of Veterinary Microbiology, University of Saskatchewan, Saskatoon, SK, CanadaSchool of Public Health, University of Saskatchewan, Saskatoon, SK, CanadaDepartment of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, T6G 2R1 Canada

r t i c l e i n f o

rticle history:eceived 5 February 2010eceived in revised form

a b s t r a c t

We generated poly[di(carboxylatophenoxy)-phosphazene] (PCPP) microparticles encapsulating ovalbu-min (OVA) and CpG of 0.5–2.5 �m in diameter with an encapsulation efficiency of approximately 63%and 95% respectively. In mice the microparticles generated high antigen-specific IgG, IgG1 and IgG2a

0 September 2010ccepted 26 September 2010vailable online 29 October 2010

eywords:olyphosphazeneicroparticles

titers with higher IgG2a/IgG1 ratios. Whole body in vivo imaging of mice subcutaneously injected withMPs showed several fold increase of OVA and CpG in draining inguinal lymph nodes compared to solubleformulations. We conclude that PCPP MPs are more effective in enhancing immune responses comparedto soluble formulations, due to co-delivery of OVA and CpG resulting in a Th1 type of immune response.

© 2010 Elsevier Ltd. All rights reserved.

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. Introduction

Vaccine formulations are made up of three critical components:1) antigen against which potent adaptive immune responses areesired, (2) an adjuvant which can stimulate an innate immuneesponse, and (3) a delivery vehicle for optimal presentation to bothnnate and adaptive immune systems. Alum is the most widely useddjuvant approved for use in humans. However, alum has severalndesirable side-effects including a significant inflammatory reac-ion at the site of injection, a strong bias towards a Th2 response,

inimal induction of cell mediated immunity, short duration ofmmunity, and the need for repeated booster immunizations [1–3].ence, novel adjuvants are currently being sought that will pro-

ote strong humoral and cellular immunity, balanced T helper

(Th1)/Th2 immune cell response and a long duration of immunityith minimal side effects. Furthermore, the formulation should also

e biodegradable and economical.

∗ Corresponding author at: Vaccine and Infectious Disease Organization (VIDO),niversity of Saskatchewan, 120 Veterinary Road, Saskatoon S7N 5E3, SK, Canada.el.: +1 306 966 1513; fax: +1 306 966 7478.

E-mail address: volker.gerdts@usask.ca (V. Gerdts).1 These authors contributed equally.

264-410X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2010.09.080

CpG when used as an adjuvant, has demonstrated to be potentinfluencers of Th1 responses in mice against tumor antigens andSalmonella [4–8]. While CpG exhibits potent immunostimulatoryeffects, the rapid degradation and ineffective delivery into the intra-cellular compartments of antigen presenting cells (APCs) are majorobstacles in improving its efficacy [9]. Co-administration of antigenand CpG produces a potent Th1 shift which is required for killingof intracellular pathogens like hepatitis B virus and Mycobacterium[10–13]. These studies highlight the fact that delivery of the anti-gen and CpG at the same time significantly enhanced the immuneresponse in contrast to delivery of the CpG after the administrationof the vaccine. Delivery devices that protect CpG from enzymaticdegradation and improve targeting to endolysomes ensure thatboth CpG and antigen are co-delivered to the same APC, and pro-vide long term immunity [14,15]. An ideal method of deliveringCpG along with antigen may be chemically cross-linking CpG withantigen [16], however this process involves additional processingsteps which might affect the stability of the antigen. Several animalmodels using CpG in conjunction with alum [17,18], biodegrad-

able microparticles, nanoparticles [19], and liposomes [20] haveshown that combinations of adjuvants can synergistically enhanceimmune responses. To this end biodegradable and biocompati-ble polymers such as PLA (poly-lactic acid), PGA (poly-glycolicacid) and PLGA (poly{d,l}-lactide-co-glycolic acid), starch, alginate

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nd chitosan have been extensively studied for microparticle andanoparticle vaccine delivery [21]. However, the methods usedo generate particles typically use a double emulsion method torepare a water-in-oil emulsion followed by solvent evaporation.hese systems are sub-optimal for use in biological systems dueo the required exposure of antigens and adjuvants to organic sol-ents and harsh conditions like high temperatures and mechanicaltirring resulting in low encapsulation efficiency [19].

PCPP [poly(di(sodium carboxylatophenoxy)-olyphosphazene)] is a new class of ion cross-linkable,ater-soluble ionotropic polyphosphazene polymers. PCPPas been shown to act as a strong adjuvant in the presence of aariety of viral and bacterial antigens [22–24]. Using PCPP as andjuvant has an added advantage by the ability to form hydrogelicrospheres by coacervation with monovalent salt solutions likeaCl and subsequent stabilization with divalent cations (Ca++)

25]. In this study, we describe a simple method of microparticleeneration by coacervation of a mixture of aqueous PCPP, OVArotein antigen and CpG 1826. To date, we are unaware of othertudies in which CpG-ODN is encapsulated in PCPP microparticles.n vitro and in vivo immunogenic studies with PCPP microparticlesMPs) co-delivering OVA and CpG were compared with solubleCPP–OVA–CpG formulations.

. Materials and methods

.1. PCPP

PCPP polymer was synthesized at Idaho National LaboratoryIdaho Falls, ID, USA) according to Andrianov et al. [26]. The poly-

er was dissolved in Dulbecco’s PBS (Sigma–Aldrich, MO, USA) byentle shaking for 36 h at room temperature (RT).

.2. Encapsulation of OVA and CpG into microparticles byoacervation

PCPP microparticles were generated by coacervation withodium chloride (NaCl) and subsequent stabilization with calciumhloride (CaCl2) as described [27] with minor modifications. Briefly,ml of 0.2% PCPP (w/v) dissolved in Dulbecco’s PBS (130 mM NaCl,.5 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4 Sigma) was mixed with00 �l of 0.25% aqueous chicken ovalbumin (OVA) grade V (Sigma)nd 50 �l of 10 mg/ml CpG 1826 oligodeoxynucleotide (Merial, GA,SA). To this mixture, 1.9 ml of 6.2% (w/v) aqueous NaCl was addedrop-wise and incubated at RT for 20 min. The coacervate was intro-uced slowly into 200 ml of 8.8% (w/v) CaCl2 solution and stirredor 10 min. MPs were isolated by centrifugation at 1340 × g at RTn a Sorvall LEGEND® centrifuge (Kendro Laboratories, NC, USA)nd washed with deionized water. MPs were either stored in PBSt 4 ◦C or were frozen at −80 ◦C for at least 1 h and lyophilized for4 h in a Labconco lyophilizer (Labconco Corporation, MS, USA) in

yophilization vials. The efficiency of antigen encapsulation wasetermined by agitating the particles in 10× PBS (pH 7.4) on aUTATOR® (TCS Scientific Corp., PA, USA). OVA and CpG were quan-

ified using QUANTI-ITTM protein and QUANTI-ITTM ssDNA assayits (Molecular Probes, CA, USA), respectively. The encapsulationfficiency of the MPs was estimated as E = (total amount of proteinn particles/total amount used) × 100 and the loading capacity wasstimated as L = (weight of protein encapsulated/weight of parti-les) × 100.

.3. Microparticle size and morphology

To establish the microparticle size, morphology and the effi-iency of antigen and adjuvant encapsulation, MPs were lyophilizednd size fractionation done by Coulter Counter (Beckman

8 (2010) 8306–8314 8307

COULTER® Inc., Fullerton, CA, USA). The lyophilized microparti-cle, soluble and aqueous formulations were observed under aScanning Electron Microscope (SEM) (JM4500, JEOL Ltd., Tokyo,Japan) at an operating voltage of 15 kV at 5000× magnification.MPs prepared by encapsulating FITC-OVA and ALEXAFLUORTM-594 labeled CpG 1826 (Molecular Probes, CA, USA) were observedunder a confocal laser scanning microscope (CLSM) using ZeissLSM410 at the Saskatchewan Structural Sciences Centre, Univer-sity of Saskatchewan, using neon argon laser. Images were capturedand analyzed by LSM410 and ImageJ software.

2.4. Microparticle release kinetics of OVA and CpG

Five milligrams of MPs encapsulating OVA, CpG alone, or co-encapsulating OVA plus CpG were suspended in 5 ml of PBS. Thesolutions were shaken on an orbital shaker at 200 rpm at 37 ◦C andsupernatants were collected at defined time points. The concentra-tion of CpG and OVA released in the supernatants were estimatedby spectrophotometry at 260 and 280 nm, respectively.

2.5. Microparticle uptake analysis in macrophages

Mouse J774.1 macrophages obtained from ATCC were main-tained in complete Dulbecco’s Modified Eagles medium (DMEM)supplemented with 10% FBS and antibiotics at 37 ◦C and 5% CO2.The cells were sub-cultured every 2–3 days by vigorous pipet-ting and/or cell scraping in serum free media. Cell viability wasestimated by Trypan Blue exclusion. The cells were plated in 24well cell culture plates at 1 × 106 cells/ml in DMEM media. MPsencapsulating FITC-OVA and/or CpG were overlaid in each well intriplicate and incubated as above for 1 and 2 h. The cells were har-vested, washed with FACSFLOW (BD Biosciences), and sorted on aFACSCALIBURTM flow cytometer (Beckton-Dickinson, NJ, USA) andthe data was analyzed with CELLQUESTTM software. For CLSM stud-ies macrophages grown in 8 well tissue culture chamber slides andincubated with fluorescent MPs encapsulating ALEXAFLUORTM-488 OVA at 37 ◦C, 5% CO2, washed with sterile PBS and thenincubated in 5 nM LYSOTRACKER® Red (Molecular probes, CA, USA)in complete media for 30 min and again washed three times withPBS. The chambers were then removed and fixed in 100% methanolat RT for 15 min and dried in the dark. The cells were overlaid witha drop of PROLONG® GoldTM anti-fade agent (Molecular Probes)and a coverslip was placed over the drop to let the anti-fade agentspread over the entire surface. The slides were allowed to cure for24 h in the dark at RT and then they were observed under the con-focal microscope. A representative cell was selected and a series ofoptical sections (z-sections) were taken in dual filter mode. Imagescaptured in RITC, FITC and bright field were over-laid to determinethe lysosomal localization of fluorescent particles.

2.6. Macrophage stimulation studies using MPs

J774.1 macrophages were plated in 24 well plates with1 × 106 cells/ml in complete media. With MPs co-encapsulatingOVA (MP-OVA), CpG (MP-CpG), or OVA and CpG (MP-OVA-CpG)in PCPP and incubated for 48 h. ELISAs for TNF-�, IL-6 and IL-12were performed on the supernatants using DUOSET® ELISA kits(Research and diagnostics systems, USA) as per the manufacturer’sinstructions.

2.7. Whole body imaging of mice

5′ amino-modified CpG 1826 and OVA were labeled with LI-COR® fluorescent IRDYE® 800CW and LI-COR fluorescent IRDYE680, respectively, as per the manufacturer’s instructions with thefollowing modifications. Briefly, 1 mg of 5′-amine-modified®CpG

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308 S. Garlapati et al. / Va

826 was resuspended in 1 ml of PBS (pH 8.5) and mixed with 1ube of dye dissolved in 25 �l ultrapure water for 2 h at RT in theark. The unlabeled dye was separated from the labeled productsy centrifugation at 4000 × g for 10 min in a 4 ml YM3 MICROCON®

Millipore, MA, USA) to separate unconjugated dye from the labeledroducts. All animal experiments were carried out in accordanceith the Guidelines for the Care and Use of Laboratory Animals

s indicated by the Canadian Council on Animal Care and theniversity of Saskatchewan. At least one week prior to injection,ALB/c mice were placed on AIN-93G Purified Diet (Harlan Teklad,I, USA) to eliminate potential interference from fluorescence

n standard pelleted diets. The ventral surfaces of the mice werehaved prior to injection. To evaluate the efficacy and distributionf the components of the vaccine formulation, mice were inocu-ated subcutaneously in the gluteal region with 30 �l containing0 �g 680-OVA (OVA-680), 10 �g 800CW-CpG 1826 (CpG-800) orCPP as indicated. Control mice were injected with either saliner hydrolyzed 680/800CW IR Dye (carboxylate form) to act as aoint of reference for background fluorescence and unconjugatedye, respectively. Vaccines were co-formulated with OVA-680 andpG-800 as soluble formulation or MPs. Mice were scanned at 2 h,4 h and 7 days post-injection. To maintain sedation during the

maging process, the mice were anesthetized with 2% isoflurane.maging was performed using the ODYSSEY® Imaging System and

ousePOd® (LI-COR, NE, USA), which allowed for administrationf anesthesia and maintenance of localized temperature of 37 ◦Co reduce stress on the mice. At each time point, mice were sacri-ced by cervical dislocation and the draining inguinal lymph nodesere surgically removed and snap-frozen in liquid nitrogen. Lymphodes were stored in a 96 well IMMULON® plate and stored at80 ◦C until scanning.

.8. Mouse immunization studies

Fifty-six BALB/c mice (Charles River) (n = 8 per group) werenjected subcutaneously with OVA alone (AQ-OVA), OVA-CpG aloneAQ-OVA-CpG), OVA encapsulated in MP (MP-OVA), OVA and CpGncapsulated in MPs (MP-OVA-CpG), OVA with PCPP (SOL-OVA),VA and CpG with PCPP (SOL-OVA-CpG) and PBS (negative con-

rol) in 50 �l on the back. The aqueous (AQ) formulations did notnclude PCPP, the soluble formulation (SOL) includes PCPP and the

P formulations are also with PCPP. The amount of OVA, CpG andCPP used were 20 �g, 10 �g, and 50 �g where applicable. Miceere immunized on day 0 and a booster immunization was given

n day 28, and blood was collected using tail puncture on day 0, 2,, 6, 8 weeks.

.9. Detection of OVA specific antibodies by ELISA

IMMULON® II microtiter plates (Dynex Technology Inc., VA,SA) were coated overnight at 4 ◦C with OVA at 10 �g/ml in car-

ig. 1. Scanning electron micrograph of (A) MP-OVA-CpG, (B) SOL-OVA-CpG and (C) AQ-000× magnification. The inset scale bar corresponds to 1 �m for all the figures.

8 (2010) 8306–8314

bonate coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6) and100 �l of antigen was added to each well. Wells were washed 6times with Tris buffered saline (pH 7.3) containing 0.05% TWEEN®

20 (TBST). Diluted mouse serum samples were added to the wells at100 �l/well and incubated for 1 h at room temperature. Wells werewashed again with TBST and biotinylated goat-anti mouse IgG, IgG1and IgG2a antibodies (Caltag Laboratories, CA, USA) were added towells at 100 �l/well and incubated for 1 h at RT. Wells were washedand alkaline phosphatase conjugated with streptavidin (CedarlaneLaboratories, ON, Canada) diluted 1/5000, was added in each wellfollowed by 1 h incubation at RT. Wells were washed 8 times indouble distilled water (ddH2O). Di(Tris) p-nitrophenyl phosphate(PNPP) (Sigma) was diluted 1/100 in PNPP substrate buffer (1 mMof MgCl2, 200 mM of Tris–HCl, pH 9.8) and 100 �l/well was added.The reaction was allowed to develop for 15 min, and absorbancewas read as optical density (OD) at 405 nm in a Microplate Reader(Bio-Rad Laboratories, Hercules, CA). Results are reported as titers,which are the reciprocal of the highest dilution that gave a positiveOD reading. A positive titer was defined as an OD reading that was atleast two times greater than the values for a negative sample. Neg-ative samples were represented by naive, non-immunized mice.

2.10. Statistical analysis

All data on IgG, IgG1 and IgG2a, antibody titers in BALB/cmice were analyzed using GRAPHPAD PrismTM version 5.01for Windows®, (GraphPad Software, San Diego, California, USA,www.graphpad.com) and STATISTIXTM 7.0 software. The meanserum titers from ELISAs were examined for significance usingrepeated measures ANOVA with Tukey’s Comparison of Rank Sum.Data from cytokine ELISAs from cell culture supernatants wereexamined using the Kruskal–Wallis test. If the means were foundto be significant, median ranks between pairs of groups wereperformed using two-tailed Mann–Whitney U-tests. Mean com-parisons were conducted to compare the magnitude of responses.Significant effects were declared at p < 0.05.

3. Results

3.1. Microparticle morphology, size distribution andencapsulation efficiency

The size distribution analysis using coulter particle size ana-lyzer showed that more that 90% of the MPs were between 0.5 and2.5 �m in diameter. SEM studies using lyophilized MPs confirmedspherical morphology between 0.5 and 2.5 �m (Fig. 1A) while in

the soluble formulations the particles were smaller than 100 nm(Fig. 1B) and aqueous formulations showed larger particles lackingspherical morphology (Fig. 1C). The encapsulation efficiency wasestimated to be between 66 and 70% for OVA, 95% for CpG and theloading capacity was estimated to be 2.6 mg/g.

OVA-CpG. The formulations were freeze dried lyophilized for 24 h and observed at

S. Garlapati et al. / Vaccine 28 (2010) 8306–8314 8309

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ig. 2. Confocal laser scanning microscopic images of FITC-OVA/ALEXAFLUOR-546he middle panel shows red fluorescent which represents CpG-1826-ALEXAFLUORppears as two, closely associated layers with CpG-1826-Alexa 594 dispersed withinhe reader is referred to the web version of the article.)

Confocal imaging of MP-OVA-CpG entrapping FITC OVA andLEXAFLUOR-594 labeled CpG ODN using green and red channelshowed that OVA localized to the surface of the MPs in two layersFig. 2A), while CpG was encapsulated inside the microparticles indispersed pattern (Fig. 2B).

The stability of the MPs at physiological conditions (37 ◦C, pH.4) and the kinetics of release of OVA and CpG in vitro fromP-OVA and PCPP and MP-CpG were measured spectrophotomet-

ically with an increase in absorbance at 260 nm corresponding toncreased release of OVA from the MPs and absorbance at 280 nmorresponding to increased release of CpG from the MPs. At phys-ological conditions, the release of OVA and CpG occurred in threehases and were released in parallel (Fig. 3). The initial peak wasbserved at 1 h followed by a sustained release and a third peakfter about 500 h (21 days).

.2. PCPP MPs are efficiently phagocytosed by J774 macrophages

The ability of murine J774 macrophages to phagcytose MPs wasetermined by overlaying J774 macrophages with MP-OVA-CpGncapsulating FITC OVA (cell particle ratio of 2:1) and estimatinghe cell-associated fluorescence intensity by FACS cytometric anal-sis (Fig. 4). Data are presented as a scatter plot of forward angleight scatter (FALS; y-axis) versus log10 fluorescence intensity (x-xis). Untreated live cells gated for high granularity were screened

or auto fluorescence and set as the baseline (Fig. 4A and B). Afterncubation for 1 h at 37 ◦C (energy-dependent endocytosis), morehan 53% of MPs were found to be cell-associated (Fig. 4D) and,ithin 2 h, 98% of the gated cells showed increase in fluorescence

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ig. 3. The release kinetics of OVA from MP-OVA or CpG from MP-OVA-CpG incu-ated at 37 ◦C. MPs encapsulating OVA or CpG 1826 were incubated at 37 ◦C in PBSn a stirrer at 200 rpm. Absorbance was measured at 260 nm for OVA and 280 nmor CpG-1826 at various time points (n = 2). There was an initial burst release of OVAnd CpG 1826 and then the absorbance values came down up to about 50 h. Afterhich, there was sustained release of both particles from 50 to 500 h where the

elease peaked. The results were consistent in at least 2 independent experiments.

826/PCPP MP. The left panel shows green fluorescent which represents FITC-OVA,nd the right panel shows the overlay of the green and red fluorescence. FITC-OVAentre of the MP. (For interpretation of the references to colour in this figure legend,

indicating phagocytosis of MPs (Fig. 4E). At 4 ◦C, less than 2% ofthe cells (Fig. 4C) were found to be cell associated (non-specificadherence).

Intracellular localization of MPs in macrophages was investi-gated using CLSM. After the macrophages were incubated withgreen fluorescent MP for 2 h, the cells were later incubated withLYSOTRACKER Red, a fluorescent acido-tropic probe for labelingand tracking acidic organelle like lysosomes in live cells. Thereforeif the green fluorescent MPs are indeed in the red labeled lysosomalcompartment they should fluoresce both green and red at the sameplane i.e., in the same location. A series of such z-sections showinggreen florescent MPs (Fig. 5A) were over-laid at the same plane forred fluorescence for labeled lysosome (Fig. 5B) and the overlay con-taining both red and green fluorescence appeared yellow (Fig. 5D).Hence it could be concluded that the MPs are indeed internalizedwithin the lysosomes.

3.3. MP-OVA-CpG encapsulating OVA and CpG elicitpro-inflammatory cytokines

Macrophage cells (1 × 106) were treated with OVA and CpG inaqueous solutions, without PCPP (AQ), soluble with PCPP (SOL), andPCPP microparticles (MP) formulations with 10 �g OVA, 2.5 �g CpGand 25 �g PCPP as appropriate. The cells were cultured at 37 ◦Cand 5% CO2 for 48 h and cell culture supernatants were testedusing sandwich ELISA. TNF�, IL-6, IL-12p70 levels were signifi-cantly higher in macrophages treated with MP compared to withSOL and AQ formulations. There was no production of cytokinesobserved in macrophages treated with empty MP, or MP with OVAalone (results not shown) (Fig. 6).

3.4. Systemic distribution of PCPP microparticles and solubleformulations in mice

Whole animal imaging was performed on BALB/c miceinjected subcutaneously with saline, inactive forms of 680 and800 CW IRDYE (carboxylate dyes), OVA labeled with 680 IRDYE(OVA-680), CpG 1826 labeled with 800CW IR dye (CpG-800),and 50 �g PCPP as appropriate. Mice were scanned using theODYSSEY Imaging System, MOUSEPOD [28]. Fig. 7A shows imagesof representative mice taken at 2 h, 2 days, and 7 days post-dosing. Two hours post-injection, high fluorescent intensity wasobserved throughout the entire body in mice injected with carboxy-late dyes (Fig. 7A(b)), or SOL-OVA-CpG (Fig. 7A(c)) whereas miceinjected with MP-OVA-CpG (Fig. 7A(d)) showed very little migra-tion of labeled antigen or adjuvant from the site of injection. Mice

injected with saline alone showed only background fluorescence(Fig. 7A(a)). Mice injected with carboxylate dyes showed signifi-cant dispersal of dyes throughout the body after 2 days and, to alesser extent, after 7 days. High fluorescent intensity was observedat the injection site after 7 days in mice injected with SOL formula-

8310 S. Garlapati et al. / Vaccine 28 (2010) 8306–8314

Fig. 4. Flow cytometric data of J774 macrophages (1 × 106) incubated with of fluorescent MP-OVA-cpG at 2:1 ratio of MP:macrophage showed that more than 53% of them ocytos es forp the boi

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icroparticles are incorporated by the end of 1 h, 99% in 2 h (energy mediated endpecific adherence). The upper left panel shows the gating of untreated macrophaganel shows cells treated with fluorescent microparticles but incubated at 4 ◦C and

ncubated with MP for 2 h.

ion (Fig. 7A(c)) however the signal was significantly higher in theP formulation (Fig. 7A(d)).The fluorescent intensity of OVA-680 and CpG-800 from drain-

ng lymph nodes was compared across all animal groups 2 days anddays post-injection (Fig. 7B). Each channels’ fluorescent intensity

s represented as fold change relative to mice injected with PBS.he inguinal LNs of mice injected with the MP-OVA-CpG formu-ation showed a higher retention of both OVA-800 and CpG-800ays and 7 days post-injection as compared to SOL-OVA-CpG. Theseata indicate that the MP formulation appeared to reduce the sys-emic distribution of labeled OVA and CpG, and significant OVAnd CpG were present in the lymph nodes 2 days and 7 daysost-injection.

.5. Antigen-specific antibody responses in serum: MPsncapsulating OVA and CpG show high IgG2a antibody titers

To further define the ability of MPs to enhance antigen-specificntibody responses in vivo, mice were immunized subcutaneously

ig. 5. Localization of FITC OVA encapsulating MP-OVA-CpG in murine macrophage cellreatment with dye and a series of optical z sections were taken in dual filter mode. Imocalization of fluorescent-particles in the lysosomes of macrophages. The representative

acrophage in monochromatic light (C); and an overlay of lysosomes and phagocytosed M�m. (For interpretation of the references to colour in this figure legend, the reader is re

sis). In contrast, only 1.97% particles were taken up in cells incubated at 4 ◦C (non-high granularity; the middle top panel shows gated untreated cells. The top rightttom left panel shows cells treated with MP for 1 h and the right panel shows cells

with AQ-OVA, AQ-OVA-CpG, MP-OVA, MP-OVA-CpG, SOL-OVA,SOL-OVA-CpG and assayed for OVA-specific antibody titers in theserum. Total antigen-specific IgG titers in the mice treated withSOL-OVA-CpG or MP-OVA-CpG, formulation groups were signifi-cantly higher when compared with other groups but not from eachother after 6 or 8 weeks.

IgG1 titers followed a trend similar to that seen with total IgG at8 weeks such that immunization with SOL-OVA-CpG and MP-OVA-CpG induced significant (p < 0.05) increases in IgG1 titers comparedto other groups but they were not significantly different from eachother (Fig. 8C). At 6 weeks, SOL-OVA-CpG and MP-OVA-CpG wereshown to induce significant (p < 0.05) IgG1 titers compared to allgroups except SOL-OVA which also showed significant (p < 0.05)induction of OVA-specific IgG1 titers.

Immunization of mice with MP-OVA-CpG formulations showedsignificant induction of IgG2a titers after 6 weeks compared to othergroups but no significant difference was observed between othergroups. However, at 8 weeks, there was a significant rise in MP-OVA-CpG induced IgG2a anti-OVA antibody production compared

line J774 in vitro using LYSOTRACKER Red. A representative cell was imaged afterages captured in RITC, FITC and bright field and were overlaid to determine co-macrophage shows FITC MP-OVA-CpG in green (A); labeled lysosomes in red (B);P-OVA-CpG inside the lysosomes (yellow) (D). The scale bar in the inset represents

ferred to the web version of the article.)

S. Garlapati et al. / Vaccine 28 (2010) 8306–8314 8311

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ig. 6. Cytokine levels estimated by ELISA using J774 macrophages (1 × 106) were tr with MP-OVA-CpG at 37 ◦C, 5%CO2 in triplicate. Statistical significance between et p < 0.05.

o that observed in the same animals at 6 weeks and compared toll other groups (p < 0.05) (Fig. 8C).

We then calculated the IgG2a/IgG1 ratios as a way of evaluat-ng whether Th1 or Th2 type immune responses were induced byifferent formulations. Mean values of IgG2a titers up to 8 weeksfter immunization showed that all formulations produced a Th2iased immune response up to 4 weeks post-immunization. Afterhis time, only the mice immunized with MP-OVA-CpG formula-ion showed a ratio of 1.0 signifying a balanced Th1/Th2 responsend this response shifted further to approximately 1.5-fold afterweeks suggesting a predominantly Th1 type immune response

Fig. 8D) (p < 0.05). These data indicate that MP-OVA-CpG for-ulation promotes a stronger Th1 type immune response than

OL-OVA-CpG or vaccine made up with AQ-OVA and/or AQ--OVA-pG.

. Discussion

Use of multi-component vaccine adjuvants require delivery ofll components including antigen to the antigen presenting cellso develop synergistic innate and adaptive immune responses. Were interested in using polyphosphazenes and CpG as adjuvants to

enerate and enhance potent antigen specific immune responses.ater soluble polyphosphazene PCPP has been extensively inves-

igated as an immuno-stimulant and showed promising evidencen clinical trials [26,29,30]. While both polyphosphazenes and CpGave been reported to be potent adjuvants, polyphosphazene–CpG

with 10 �g OVA and 2.5 �g CpG alone (AQ-OVA-CpG) or with PCPP SOL-OVA-CpGir of groups were tested using Mann–Whitney U-test and reported to be significant

combination has been found to be more effective in enhancingimmune responses than CpG alone [31,32]. A variety of strategieshave been currently described [21,33] for co-delivery of antigenand CpG to the same antigen presenting cell to promote a syn-ergetic immune response. When CpG was delivered in PLG MPsencapsulating meningococcus B antigen in soluble, adsorbed andencapsulated formulations the encapsulated CpG enhanced func-tional antibodies by more than 4 times [34]. Liposomes havealso been used for the delivery of CpG resulting in an enhancedTh1 immune response inducing protection against a variety ofpathogens [20,35,36]. Nanoparticles containing CpG, when deliv-ered to the lymph nodes provided efficient anti-tumor activity [37].In addition, CpG microparticles delivered to mucosal routes pro-vided protection against malaria [38,39] hepatitis [39] and HIV [40]in mice. Clearly, MP as a delivery vehicle for CpG has shown toprovide a broad spectrum of beneficial immune responses.

In the present study, we report encapsulation of CpG and OVA inPCPP-based microparticles using a simple coacervation with NaCl[25] which results in MP sizes within the range preferred by antigenpresenting cells for internalization [41,42]. The size and morphol-ogy of the MPs has an important bearing on the immune response asdescribed earlier [43,44]. When MPs of size 0.5 �m and 3.9 �m were

used to immunize animals with tetanus toxoid antigen, the latergave higher antibody responses than the former. It has also beenwell described that MPs less than 10 �m are preferentially up takenby APCs [45]. We observed greatly facilitated MP uptake and morepotent effects on immune responses than previously reported. This

8312 S. Garlapati et al. / Vaccine 2

Fig. 7. Real-time imaging of near-infrared dye fluorescence on the ventral surfaceof BALB/c mice post-inoculation. Fluorescent images were obtained following asubcutaneous injection of saline (a), 680CW and 800 carboxylate dyes (b), 20 �gOva-680, 10 �g CpG 800 and PCPP in a MP (c) or soluble (d) formulation. Controlanimals (a, b) were injected with either saline or hydrolyzed 680/800CW IRDye(carboxylate form) to act as a point of reference for background fluorescence andunconjugated dye, respectively. (A) Whole-body images were obtained 2 h, 24 h,and 7 days post-injection using ODYSSEY Imaging System under identical imagingconditions. The first panel in each time point shows a combination of the 680 nmand 800 nm signals while the second and third panels represent heat-maps ofthe 680 nm and 800 nm channels, respectively. Intensity and sensitivity settingswere held constant for each image. Red indicates the highest intensity, bluerepresents the lowest intensity. The baseline intensity and sensitivity settingsfor imaging were held constant in each scan. In each set of images, there are3 panels. The top panel represents mice scanned at 680 nm (red fluorescence)and 800 nm (green fluorescence) which measures OVA and CpG, respectively.Both the red and green channels are shown together in this panel only. Themiddle panel is a false-colour image representation of the 680 nm channel andthe bottom panel is a false-colour image representation of the 800 nm channel.These false-colour images map the intensity of fluorescence to a colour palletwhich is shown as a legend on the extreme right of the image. Any red in thefalse-colour images does not represent the 680 nm wavelength but instead itrepresents the intensity of either the 680 nm or 800 nm fluorescence. Since the800 IRDYE is scanned at 800 nm, there is very little background fluorescence fromfeed and/or body tissues. The 680 IRDYE is scanned at 680 nm which does competewith some natural fluorescence of the body tissues and ultimately, it has a higher

8 (2010) 8306–8314

method generated particles with high encapsulation efficiencies ofCpG that were nearly 3-fold higher than as previously reported aswell as 2-fold with respect to OVA [19]. This high encapsulation can,in part, be explained due to the formation of water-soluble non-covalent protein-polymer complexes [46] which better facilitatesencapsulation by coacervation, which is normally not observedwith other water-insoluble polymer adjuvants like PLGA [19]. Highencapsulation efficiencies of PCPP microparticles might further beexplained by mild encapsulation conditions used here as comparedto other methods where the protein antigen and CpG are requiredto be exposed to organic solvents like di-chloromethane, poly-vinylalcohol and subjected to high stirring speeds for prolonged timewhich might have a role in their degradation during particle syn-thesis [19]. CLSM revealed that OVA was present on the surface ofthe particles and CpG was encapsulated inside the microparticles,which may protect CpG from degrading nucleases in vivo. The CLSMstudy convincingly showed the encapsulation of CpG and antigento physically distinct locations of the MP and hence will not haveany effect on physio-chemical properties of either OVA or CpG.

Encapsulating CpG and antigen in a polyphosphazene-basedmicroparticle revealed specific advantages over soluble vaccineformulations: (1) MP-OVA-CpG enhanced immune responses com-pared to SOL-OVA-CpG formulations, (2) IgG, IgG1 and IgG2aantibody titers were higher in formulations composed of MP-OVA-CpG compared to SOL MP-OVA-CpG and (3) addition of CpG had astrong influence in generating a statistically significant IgG2a anti-body response, MP-OVA-CpG compared to SOL-OVA-CpG changedthe IgG2a:IgG1 ratio to 1.5 denoting a clear Th1 shift in theimmune response when formulated in MPs. This enhanced Th1response is essential for counteracting intracellular pathogens suchas Mycobacterium sp. and Hepatitis B virus [11] and clearance oftumors [47]. Hence, we conclude that MPs co-encapsulating OVAand CpG are able to induce a change in the quality of immuneresponses from a natural Th2 response for OVA to a clear Th1suggesting significant potential in a wide variety of immunothera-peutic and prophylactic applications.

Reasons for MP-OVA-CpG promoting such a significantlystronger Th1 type immune response may be due to the particu-late nature and release kinetics of the antigen/adjuvant within theMP. In vitro release kinetic studies revealed an initial burst of anti-gen/adjuvant release followed by both a sustained and a terminalburst release, SEM indicated that MP-OVA-CpG formulations werehighly particulate consisting of different sizes of MPs which wespeculate may be responsible for the release patterns of the anti-gen/adjuvant (Fig. 3). We speculate that particles of different sizesare degraded at different times resulting in pulsatile release. Thedegradation of MPs may be explained by the breakdown proper-ties of PCPP which has been shown by a time dependent releaseof the side groups followed by breakdown of the polymer chainat physiological conditions [48,49]. Hence we speculate that thesmaller particles which contain lower amount of PCPP degradefaster than larger particles. Most of the other studies on releasekinetics using poly-lactide-co-glycolic acid (PLGA) microparticlesshowed an initial burst followed by a sustained release but not a

pulsatile release mainly due to uniform particle size [19]. Interest-ingly, the large release at 3 weeks coincides with a routine regimenof administrating a 2nd immunization at 3–4 weeks after initialimmunization. This finding is important because it has implicationsin the development of a single shot vaccine without the need for

background. (B) Quantification of fluorescent imaging of inguinal lymph nodes 2 and7 days post-injection using the ODYSSEY Imaging System under identical imagingconditions. Raw intensity in pixels were obtained for each whole-body image at theinjection site and compared to control mice using a consistent surface area. (Forinterpretation of the references to colour in this figure legend, the reader is referredto the web version of the article.)

S. Garlapati et al. / Vaccine 28 (2010) 8306–8314 8313

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Fig. 8. Iso-typing of anti-ova specific antibodies for mice vaccinated with SOL and MP formulations as shown in the legend above. The mice were vaccinated on day 0 andboosted on day 28. Total IgG titers were not significantly different form each other between all the groups (A). Though the IgG1 titers were slightly higher in MP-OVA-CpGg formg s of thf

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roup but they were not significantly different from groups which received solubleroups (C). And the ratio of IG2a/IgG1 were highest in than 1 MP-OVA-CpG at 8 weekrom all other groups.

ooster immunizations. MPs have not been reported before as a sin-le shot vaccine and this exciting result would have great benefitarticularly in developing countries where compliance to immu-ization schedules are often very poor.

Detailed knowledge of how PCPP-based MPs mediate theirmmune-enhancing effects are currently unknown at the mech-nistic level but will drive the development of improved vaccines.he Th1 immune response that is observed is likely due to signif-cantly higher expression of pro-inflammatory cytokines TNF-�,L-6 and IL-12 produced in macrophages treated with MP-OVA-pG compared to SOL-OVA-CpG or AQ-OVA-CpG. Release of theseytokines promotes APC maturation as well as activation of its MHClass I and II processing machinery which will result in stronger Tell activation.

In vivo whole body scanning of mice injected with OVA and CpGabeled with contrasting infra-red dyes in MP and SOL formula-ions showed OVA and CpG in the latter diffused throughout theody within 2 h after administration. As such the MP formulationslowed the systemic distribution [50] of CpG and simultaneouslyargeted CpG to the LNs. Bourquin et al. [37] also showed thatn contrast to free CpG, CpG delivered by gelatin nanoparticles,voided the systemic effects and are selectively targeted to the APCso the LNs where they mediated local immune stimulation.

The mice vaccinated with MP-OVA-CpG localized the antigennd the adjuvant strongly to the site of injection. This localizationesults in enhanced antigen/adjuvant presence in the draining LNss shown in Fig. 7B. These findings may be explained by the fact thatost MP delivery systems function on the basis of efficient phago-

ytosis and transport to the lymph nodes resulting in sustainedntigen release over extended time periods, which may allow con-

inuous presentation of antigen within the local LNs. The retentionf antigen in LNs is vital for repeated stimulation of memory B cellopulation and for maintaining antibody titers over long time [51].

Polyphosphazenes are synthetic, water-soluble biodegradablend relatively inexpensive adjuvants which showed great poten-

ulations (B). The IgG2a titers in MP-OVA-CpG group were higher than all the othere study (D). The groups denoted by different sub scripts were significantly different

tial in animal trials using soluble formulations [4,52]. The easewith which MP formulations can be made appears to have greatprospects in vaccine delivery system as well. Our data demon-strates that biodegradable PCPP microparticles that entrap bothCpG ODN and antigen ensure that both components are deliveredto the lysosomal compartment of the APC. MP-OVA-CpG promotesstronger Th1 type responses than MP-OVA and SOL-OVA-CpG. Thisimproved vaccine formulation was generated under mild condi-tions, without any expensive equipment and high encapsulationefficiency making PCPP-based MP delivery vehicles an attractiveoption to decrease vaccine formulation costs. Future studies willneed to be performed to ensure safety of the formulation andwhether this formulation in essence can act as an effective “singleshot vaccine”.

Conflict of interest

The authors have no conflicting financial interests.

Acknowledgements

We gratefully acknowledge financial support from the Billand Melinda Gates Foundation and Canadian Institutes for HealthResearch through the Grand Challenges in Global Health ResearchInitiative. (www.grandchallenges.org). NE is a holder of a post-doctoral research award from Saskatchewan Health ResearchFoundation. We sincerely thank Animal Care personnel at VIDO fortheir assistance in carrying out the animal experiments. We kindly

acknowledge Dr. Hugh Townsend for his help in statistical analy-sis. We also acknowledge John R. Klaehn, Research Scientist, IdahoNational Laboratory for synthesizing the PCPP polymer. We alsoacknowledge Tom Bonli at Department of Geology, University ofSaskatchewan for the SEM.

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[51] Gray D, Skarvall H. B-cell memory is short-lived in the absence of antigen.

314 S. Garlapati et al. / Va

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